EP1857554A1 - Monoclonal and chimeric antibodies specific for human tumor necrosis factor - Google Patents

Monoclonal and chimeric antibodies specific for human tumor necrosis factor Download PDF

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EP1857554A1
EP1857554A1 EP07012626A EP07012626A EP1857554A1 EP 1857554 A1 EP1857554 A1 EP 1857554A1 EP 07012626 A EP07012626 A EP 07012626A EP 07012626 A EP07012626 A EP 07012626A EP 1857554 A1 EP1857554 A1 EP 1857554A1
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tnf
antibody
human
pathology
antibodies
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English (en)
French (fr)
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Jan Vilcek
Junming Le
Peter Daddona
Scott Siegel
David Knight
John Ghrayeb
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New York University NYU
Janssen Biotech Inc
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Centocor Inc
New York University NYU
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention in the field of immunology and medicine relates to antibodies which are specific for human tumor necrosis factor-alpha (hTNF ⁇ ); fragments, regions and derivatives thereof; and to pharmaceutical and diagnostic compositions and production, diagnostic and therapeutic methods thereof.
  • the invention further relates to nucleotide sequences encoding such antibodies, fragments and regions, as well as vectors and hosts containing such sequences, and methods thereof.
  • TNF was originally discovered in the serum of animals injected sequentially with a bacterial vaccine (bacillus Calmette-Guerin, BCG) and endotoxin ( Carswell, E.A. et al., Proc, Natl. Acad. Sci. USA 72:3666 (1975 )).
  • bacterial vaccine bacillus Calmette-Guerin, BCG
  • endotoxin Carswell, E.A. et al., Proc, Natl. Acad. Sci. USA 72:3666 (1975 )
  • TNF is a regulatory cytokine with pleiotropic biological activities. These activities include: inhibition of lipoprotein lipase synthesis ("cachectin” activity) ( Beutler, B. et al., Nature 316:552 (1985 )), activation of polymorghonuclear leukocytes ( Klebanoff, S.J. et al., J. Immunol. 136:4220 (1986 ); Perussia, B., et al., J. Immunol. 138:765 (1987 )), inhibition of cell growth or stimulation of cell growth ( Vilcek, J. et al., J. Exp. Med.
  • MHC major histocompatibility complex
  • TNF is noted for its pro-inflammatory actions which result in tissue injury, such as induction of procoagulant activity on vascular endothelial cells ( Pober, J.S. et al., J. Immunol. 136:1680 (1986 )), increased adherence of neutrophils and lymphocytes ( Pober, J.S. et al., J. Immunol. 138:3319 (1987 )), and stimulation of the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells ( Camussi, G. et al., J. Exp. Med. 166:1390 (1987 )).
  • TNF is thought to play a central role in the pathophysiological consequences of Gram-negative sepsis and endotoxic shock ( Michie, H.R. et al., Br, J. Surg, 76:670-671 (1989 ); Debets, J.M.H. et al., Second Vienna Shock Forum, p.463-466 (1989 ); Simpson, S.Q. et al., Crit. Care Clin. 5:27-47 (1989 )), including fever, malaise, anorexia, and cachexia.
  • Endotoxin is a potent monocyte/macrophage activator which stimulates production and secretion of TNF ( Kombluth, S.K. et al., J. Immunol.
  • TNF could mimic many biological effects of endotoxin, it was concluded to be a central mediator responsible for the clinical manifestations of endotoxin-related illness. TNF and other monocyte-derived cytokines mediate the metabolic and neurohormonal responses to endotoxin ( Michie, H.R. et al., N. Eng. J. Med. 318:1481-1486 (1988 )). Endotoxin administration to human volunteers produces acute illness with flu-like symptoms including fever, tachycardia, increased metabolic rate and stress hormone release ( Revhaug, A. et al., Arch, Surg. 123:162-170 (1988 )).
  • Elevated levels of circulating TNF have also been found in patients suffering from Gram-negative sepsis ( Waage, A. et al., Lancet 1:355-357 (1987 ) ; Hammerle, A.F. et al., Second Vienna Shock Forum p. 715-718 (1989 ); Debets, J.M.H. et al., Crit. Care Med. 17:489-497 (1989 ); Calandra, T. et al., J. Infec. Dis. 161:982-987 (1990 ).
  • TNF tumor necrosis factor 4 ng/kg
  • endotoxin 4 ng/kg
  • TNF tumor necrosis factor 4 ng/kg
  • Chronic intravenous TNF infusion into humans or rats was associated with anorexia, fluid retention, acute phase responses, and negative nitrogen balance (i.e., classic catabolic effects), leading to the conclusion that TNF may be responsible for many of the changes noted during critical illness ( Michie, H.R. et al., Ann, Surg. 209:19-24 (1989 )).
  • Passive immunotherapy directed at neutralizing TNF may have a beneficial effect in Gram-negative sepsis and endotoxemia, based on the increased TNF production and elevated TNF levels in these pathology states, as discussed above.
  • Yone et al ( EPO Patent Publication 0288088, October 26, 1988 ) disclosed anti-TNF antibodies, including mAbs, and their utility in immunoassay diagnosis of pathologies, in particular Kawasaki's pathology and bacterial infection.
  • the body fluids of patients with Kawasaki's pathology infantile acute febrile mucocutaneous lymph node syndrome; Kawasaki, T., Allergy 16:178 (1967 ); Kawasaki, T., Shonica (Pediatrics) 26:935 (1985 ) were said to contain elevated TNF levels which were related to progress of the pathology (Yone et al., supra ).
  • TNF ligands which can bind to monoclonal antibodies having the following epitopes: at least one of 1-20, 56-77, and 108-127; at least two of 1-20, 56-77, 108-127 and 138-149; all of 1-18, 58-65, 115-125 and 138-149; all of 1-18, and 108-128; all of 56-79, 110-127 and 135- or 136-155; all of 1-30, 117-128 and 141-153; all of 1-26, 117-128 and 141-153; all of 22-40, 49-96 or -97, 110-127 and 136-153; all of 12-22, 36-45, 96-105 and 132-157; all of both of 1-20 and 76-90; all of 22-40, 69-97, 105-128 and 135-155; all of 22-31 and 146-157; all of 22-40 and 49-98
  • anti-TNF antibodies of the present invention competitively inhibit the binding of A2 antibodies to TNF.
  • Preferred for therapeutic use are high affinity anti-TNF ⁇ antibodies, including recombinantly and hybridoma produced monoclonal and chimeric antibodies, according to the present invention, which inhibit or neutralize human TNF- ⁇ with an in vivo inhibitory dose-50 (ID50) of at least about 1 ⁇ g/ml, more preferably at least about 100 ng/ml, most preferably at least about 15 ng/ml.
  • ID50 in vivo inhibitory dose-50
  • anti-TNF ⁇ mAbs or chimeric mAbs, or TNF ⁇ -binding fragments thereof which are particularly useful in diagnostic methods for detecting human TNF ⁇ in patients suspected of suffering from conditions associated with TNF ⁇ production, including methods wherein high affinity murine and/or chimeric anti-TNF ⁇ antibodies of the present invention are contacted with biological materials from a patient and an antigen-antibody reaction detected. Also included are kits for detecting TNF ⁇ in biological fluids comprising high affinity murine and/or chimeric anti-TNF ⁇ antibodies or fragments of the present invention, preferably in detectably labeled form.
  • the chimeric antibodies of the present invention embody a combination of the advantageous characteristics of mAbs. Like mouse mAbs, they can recognize and bind to human TNF; however, unlike mouse mAbs, the "human-specific" properties of the chimeric antibodies lower the likelihood of an immune response to the antibodies, and result in prolonged survival in the circulation through reduced clearance. Moreover, using the methods disclosed in the present invention, the constant region of any desired human immunoglobulin isotype can be combined with the desired antigen combining site.
  • the present invention is directed to a high affinity mouse mAb, designated A2, which is specific for human TNF ⁇ .
  • A2 a high affinity mouse mAb
  • the antibody may be in detectably labeled form.
  • polypeptide portions of hTNF ⁇ are provided that, when bound as part of an intact TNF ⁇ molecule by antibodies or fragments specific for epitopes included in these peptide portions, inhibit or neutralize TNF ⁇ activity in vivo.
  • the epitopic amino acids do not include amino acids from residues from at least one of 11-13, 3.7-42, 49-57 or 155-157 of hTNF ⁇ (of SEQ ID NO:1).
  • a human/murine chimeric immunoglobulin chain contains a constant (C) region substantially similar to that present in a natural human immunoglobulin, and a variable (V) region, preferably non-human, having high affinity and the desired specificity for a TNF epitope.
  • the invention also provides antibodies, fragments, and regions having chimeric H and L chains associated so that the overall molecule exhibits the desired antigen recognition and binding properties.
  • the present invention is also directed to a chimeric antibody comprising two light chains and two heavy chains, each of the chains comprising at least part of a human constant region and at least part of a variable (V) region of non-human origin having specificity to human TNF ⁇ , said antibody binding with high affinity to a inhibiting and/or neutralizing epitope of human TNF ⁇ .
  • the invention is also includes a fragment or a derivative such an antibody.
  • the v region is of non-human origin, most preferably of murine origin.
  • the V region is derived from, or binds epitopes of the A2 mAb.
  • a chimeric antibody which binds epitopes of the antibody designated chimeric A2 (cA2), or a chimeric human-mouse anti-TNF mAb that competitively inhibits the binding of cA2 to TNF ⁇ .
  • the chimeric antibody inhibits or neutralizes human TNF ⁇ in vivo with an ID50 of at least about 1 ⁇ g/ml , more preferably at least about 100 ng/ml, most preferably at least about 15, 30, 50, or 80 ng/ml.
  • the present invention provides anti-tissue necrosis factor (TNF) murine antibodies and chimeric murine-human antibodies, and fragments and regions thereof, which inhibit or neutralize TNF biological activity in vivo and are specific for human tumor necrosis factor-alpha (hTNF ⁇ ), which can be used for diagnostic and therapeutic purposes in subjects having pathologies or conditions associated with the presence of a substance reactive with anti-TNF antibody, in particular hTNF ⁇ , in amounts exceeding those present in a normal healthy subject.
  • Antibodies, and fragments, regions and derivatives thereof, of the present invention preferably contain at least one V region which recognizes an epitope of TNF which has inhibiting and/or neutralizing biological activity in vivo.
  • Preferred antibodies of the present invention are murine antibodies or high affinity human-murine chimeric anti-TNF ⁇ antibodies, and fragments or regions thereof, that have potent inhibiting and/or neutralizing activity in vivo against human TNF ⁇ :
  • Such antibodies and chimeric antibodies include those generated by immunization using purified recombinant hTNF ⁇ (SEQ ID NO:1) or peptide fragments thereof.
  • Such fragments include epitopes of at least 5 amino acids of residues 87-107, or a combination of both of 59-80 and 87-108 of hTNF ⁇ (as these corresponding amino acids of SEQ ID NO:1).
  • preferred antibodies, fragments and regions of anti-TNF antibodies of the present invention do not recognize amino acids from at least one of amino acids 11-13, 37-42, 49-57 or 155-157 of hTNF ⁇ (of SEQ ID NO:1).
  • TNF-mediated pathology Since circulating concentrations of TNF tend to be extremely low, in the range of about 10 pg/ml in non-septic individuals, and reaching about 50 pg/ml in septic patients and above 100 pg/ml in the sepsis syndrome (Hammerle, A.F. et al. , 1989, supra ) or may be only be detectable at sites of TNF-mediated pathology, it is preferred to use high affinity and/or potent in vivo TNF-inhibiting and/or neutralizing antibodies, fragments or regions thereof, for both TNF immunoassays and therapy of TNF-mediated pathology.
  • Such antibodies, fragments, or regions will preferably have an affinity for hTNF ⁇ , expressed as Ka, of at least 10 8 M -1 , more preferably, at least 10 9 M -1 , such as 5 X 10 8 , M -1 , 8 X 10 8 M -1 , 2 X 10 9 M -1 , 4 X 10 9 M -1 , 6 X 10 9 M -1 , 8 X 10 9 M -1 .
  • Preferred for human therapeutic use are high affinity murine and chimeric antibodies, and fragments, regions and derivatives having potent in vivo TNF ⁇ -inhibiting and/or neutralizing activity, according to the present invention, that block TNF-induced IL-6 secretion.
  • Also preferred for human therapeutic uses are such high affinity murine and chimeric anti-TNF ⁇ antibodies, and fragments, regions and derivatives thereof, that block TNF-induced procoagulant activity, including blocking of TNF-induced expression of cell adhesion molecules such as ELAM-1 and ICAM-1 and blocking of TNF mitogenic activity, in vivo, in situ, and in vitro.
  • murine mAb A2 of the present invention is produced by a cell line designated c134A.
  • Chimeric antibody cA2 is produced by a cell line designated c168A.
  • Cell line c134A is deposited as a research cell bank in the Centocor Cell Biology Services Depository, and cell line c168A(RCB) is deposited as a research cell bank in the Centocor Corporate Cell Culture Research and Development Depository, both at Centocor, 200 Great Valley Parkway, Malvern, Pennsylvania, 19355.
  • the c168A cell line is also deposited at Centocor BV, Leiden, The Netherlands.
  • c168A was deposited as of the filing date of the present application at the American Type Culture Collection, Rockville, Maryland, as a "Culture Safe Deposit.”
  • epitope is meant to refer to that portion of any molecule capable of being recognized by and bound by an antibody.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • inhibiting and/or neutralizing epitope is intended an epitope, which, when bound by an antibody, results in loss of biological activity of the molecule or organism containing the epitope, in vivo, in vitro and in situ, more preferably in vivo, including binding of TNF to a TNF receptor.
  • Preferred antibodies, fragments and regions of anti-TNF antibodies of the present invention recognize epitopes including 5 amino acids comprising at least one amino acid from amino acids residues 87-108 or both residues 59-80 and 87-108 of hTNF ⁇ (of SEQ ID NO:1). Preferred antibodies, fragments and regions of anti-TNF antibodies of the present invention do not recognize epitopes from at least one of amino acids 11-13, 37-42, 49-57 or 155-157 of hTNF ⁇ (of SEQ ID NO:1). In a preferred embodiment, the epitope comprises at least 2 amino acids from residues 87-108 or both residues 59-80 and 87-108 of hTNF ⁇ (of SEQ ID NO:1).
  • antibody is meant to include a polyclonal, or monoclonal antibody, and fragments and regions thereof, as well as derivatives thereof, which are capable of binding portions of TNF that inhibit binding to TNF receptors by TNF. Fragments include, for example, Fab, Fab', F(ab') 2 and Fv. These fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody ( Wahl et al., J. Nucl. Med. 24:316-325 (1983 )).
  • Regions of anti-TNF antibodies of the present invention include at least one of a heavy chain constant region (H c ), a heavy chain variable region (H v ), a light chain variable region (L v ) and a light chain constant regions (L c ), wherein a polyclonal, monoclonal antibody, fragments and regions thereof include at least one heavy chain variable region (H v ) or light chain variable region (L v ) which binds a portion of a TNF and inhibits TNF biological activity.
  • H c heavy chain constant region
  • H v heavy chain variable region
  • L v light chain variable region
  • L c light chain constant regions
  • the antibody is a monoclonal antibody which binds amino acids of an epitope of TNF, which antibody is designated A2, rA2 or cA2, which is produced by a hybridoma or by a recombinant host.
  • the antibody is a chimeric antibody which recognizes an epitope recognized by A2.
  • the antibody is a chimeric antibody designated as chimeric A2 (cA2).
  • the murine and chimeric antibodies, fragments and regions of the present invention comprise individual heavy (H) and light (L) immunoglobulin chains.
  • a chimeric H chain comprises an antigen binding region derived from the H chain of a non-human antibody specific for TNF, which is linked to at least a portion of a human H chain C region (C H ).
  • a chimeric L chain according to the present invention comprises an antigen binding region derived from the L chain of a non-human antibody specific for TNF, linked to at least a portion of a human L chain C region (C L ).
  • antigen binding region refers to that portion of an antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen.
  • the antibody region includes the "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding residues.
  • chimeric antibody includes monovalent, divalent or polyvalent immunoglobulins.
  • a monovalent chimeric antibody is a dimer (HL)) formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain.
  • a divalent chimeric antibody is tetramer (H 2 L 2 ) formed by two HL dimers associated through at least one disulfide bridge.
  • a polyvalent chimeric antibody can also be produced, for example, by employing a C H region that aggregates (e.g., from an IgM H chain, or ⁇ chain).
  • Antibodies, fragments or derivatives having chimeric H chains and L chains of the same or different V region binding specificity can be prepared by appropriate association of the individual polypeptide chains, as taught, for example by Sears et al., Proc. Natl. Acad. Sci. USA 72:353-357 (1975 ). With this approach, hosts expressing chimeric H chains (or their derivatives) are separately cultured from hosts expressing chimeric L chains (or their derivatives), and the immunoglobulin chains are separately recovered and then associated. Alternatively, the hosts can be co-cultured and the chains allowed to associate spontaneously in the culture medium, followed by recovery of the assembled immunoglobulin, fragment or derivative.
  • the antigen binding region of the chimeric antibody of the present invention is derived preferably from a non-human antibody specific for human TNF.
  • Preferred sources for the DNA encoding such a non-human antibody include cell lines which produce antibody, preferably hybrid cell lines commonly known as hybridomas.
  • a preferred hybridoma is the A2 hybridoma cell line.
  • the hybrid cells are formed by the fusion of a non-human anti-hTNF ⁇ antibody-producing cell, typically a spleen cell of an animal immunized against either natural or recombinant human TNF, or a peptide fragment of the human TNF ⁇ protein sequence.
  • the non-human anti-TNF ⁇ antibody-producing cell may be a B lymphocyte obtained from the blood, spleen, lymph nodes or other tissue of an animal immunized with TNF.
  • the antibody-producing cell contributing the nucleotide sequences encoding the antigen-binding region of the chimeric antibody of the present invention may also be produced by transformation of a non-human, such as a primate, or a human cell.
  • a B lymphocyte which produces anti-TNF antibody may be infected and transformed with a virus such as Epstein-Barr virus to yield an immortal anti-TNF producing cell ( Kozbor et al. Immunol, Today 4:72-79 (1983 )).
  • the B lymphocyte may be transformed by providing a transforming gene or transforming gene product, as is well-known in the art.
  • the antigen binding region will be of murine origin.
  • the antigen binding region may be derived from other animal species, in particular rodents such as rabbit, rat or hamster.
  • the second fusion partner which provides the immortalizing function, may be lymphoblastoid cell or a plasmacytoma or myeloma cell, which is not itself an antibody producing cell, but is malignant.
  • Preferred fusion partner cells include the hybridoma SP2/0-Ag14, abbreviated as SP2/0 (ATCC CRL1581) and the myeloma P3X63Ag8 (ATCC TIB9), or its derivatives (see: Hartlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988 )).
  • Marine hybridomas which produce mAb specific for human TNF ⁇ are formed by the fusion of a mouse fusion partner cell, such as SP2/0, and spleen cells from mice immunized against purified hTNF ⁇ , recombinant hTNF ⁇ , natural or synthetic TNF peptides, including peptides including 5 or more amino acids selected from residues 59-80, and 87-108 of TNF (of SEQ ID NO:1) or other biological preparations containing TNF.
  • a variety of different conventional protocols may be followed. For example, mice may receive primary and boosting immunizations of TNF.
  • TNF residues 87-108 or both residues 59-80 and 87-108 of TNF (of SEQ ID NO:1), fragments or combinations of peptides containing therein are useful as immunogens to raise antibodies that will recognize peptide sequences presented in the context of the native TNF molecule.
  • Particular peptides which can be used to generate antibodies of the present invention include combinations of amino acids selected from at least residues 87-108 or both residues 59-80 and 87-108, which are combined to provide an epitope of TNF that is bound by anti-TNF antibodies, fragments and regions thereof, and which binding provided anti-TNF biological activity.
  • Such epitopes include at least 1-5 amino acids and less than 22 amino acids from residues 87-108 or each of residues 59-80 and 87-108, which in combination with other amino acids of TNF provide epitopes of at least 5 amino acids in length.
  • antibodies of the present invention to small peptide sequences that recognize and bind to those sequences in the free or conjugated form or when presented as a native sequence in the context of a large protein are well known in the art.
  • Such antibodies include murine, murine human and human-human antibodies produced by hybridoma or recombinant techniques known in the art.
  • the amino acids of the epitope are not of at least one of amino acids 11-13, 37-42, 49-57 and 155-157 of hTNF ⁇ (of SEQ ID NO:1).
  • the cell fusions are accomplished by standard procedures well known to those skilled in the field of immunology ( Kohler and Milstein, Nature 256:495-497 (1975 ) and U.S. Patent No. 4,376,110 ; Hartlow, E. et al. , supra; Campbell, A., "Monoclonal Antibody Technology," In: Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13 (Burdon, R., et al., eds.), Elsevier, Amsterdam (1984 ); Kennett et al., Monoclonal Antibodies (Kennett et al., eds. pp. 365-367, Plenum Press, NY, 1980 ); de St.
  • Fusion partner cell lines and methods for fusing and selecting hybridomas and screening for mAbs are well known in the art (Hartlow, E. et al. , supra ; Kawamoto, T.et al., Meth. Enzymol 121:266-277 (1986 ) Kearney, J. F. et al., J. Immunol. 123:1548-1550 (1979 ); Kilmartin, J.V. et al., J. Cell Biol. 93:576-582 (1982 ); Kohler, G. et al., Eur. J. Immunol. 6:292-295 (1976 ); Lane, D.P. et al. , J. Immunol. Meth.
  • the hTNF ⁇ -specific murine or chimeric mAb of the present invention may be produced in large quantities by injecting hybridoma or transfectoma cells secreting the antibody into the peritoneal cavity of mice and, after appropriate time, harvesting the ascites fluid which contains a high titer of the mAb, and isolating the mAb therefrom.
  • hybridoma cells are preferably grown in irradiated or athymic nude mice.
  • the antibodies my be produced by culturing hybridoma or transfectoma cells in vitro and isolating secreted mAb from the cell culture medium.
  • Monoclonal antibodies of the present invention recognize epitopes including non-contiguous residues located within the non-contiguous sequences residues 87-108 or both residues 59-80 and 87-108 of TNF (of SEQ ID NO:1).
  • Preferred anti-TNF mAbs are those that inhibit this binding of human TNF- ⁇ to its receptors by virtue of their ability to bind to one or more of these peptide sequences. These antibodies would block the activity of TNF by virtue of binding to the epitope of sequences including 87-108 and/or 110-128 of TNF (of SEQ ID NO:1). Such binding is demonstrated to inhibit TNF activity, as described herein.
  • Particular peptides which can be used to screen antibodies of the present invention include combinations of amino acids selected from at least residues 87-108 or both residues 59-80 and 87-108, which are combined to provide an epitope of TNF that is bound by anti-TNF antibodies, fragments and regions thereof, of the present invention, and which binding provided anti-TNF biological activity.
  • Such epitopes include at least 1-5 amino acids and less than 22 amino acids from residues 87-108 or each of residues 59-80 and 87-108, which in combination with other amino acids of TNF provide epitopes of at least 5 amino acids in length.
  • Recombinant murine or chimeric murine-human or human-human antibodies that inhibit TNF and bind an epitope included in the amino acid sequences residues 87-108 or both residues 59-80 and 87-108 of hTNF ⁇ (of SEQ ID NO:1), can be provided according to the present invention using known techniques based on the teaching provided herein.
  • cDNA requires that gene expression elements appropriate for the host cell be combined with the gene in order to achieve synthesis of the desired protein.
  • the use of cDNA sequences is advantageous over genomic sequences (which contain introns), in that cDNA sequences can be expressed in bacteria or other hosts which lack appropriate RNA splicing systems.
  • Human genes which encode the constant (C) regions of the murine and chimeric antibodies, fragments and regions of the present invention can be derived from a human fetal liver library, by known methods.
  • Human C regions genes may be derived from any human cell including those which express and produce human immunoglobulins.
  • the human C H region can be derived from any of the known classes or isotypes of human H chains, including gamma, ⁇ , ⁇ , ⁇ or ⁇ , and subtypes thereof, such as G1, G2, G3 and G4. Since the H chain isotype is responsible for the various effector functions of an antibody, the choice of C H region will be guided by the desired effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity (ADCC).
  • the C H region is derived from gamma 1 (IgG1), gamma 3 (IgG3), gamma 4 (IgG4), or ⁇ (IgM).
  • Human immunoglobulin C regions are obtained from human cells by standard cloning techniques ( Sambrook, J. et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989 ) and Ausubel et al, eds. Current Protocols in Molecular Biology (1987-1991 )).
  • Human C region genes are readily available from known clones containing genes representing the two classes of L chains, the five classes of H chains and subclasses thereof.
  • Chimeric antibody fragments, such as F(ab') 2 and Fab can be prepared by designing a chimeric H chain gene which is appropriately truncated.
  • a chimeric gene encoding an H chain portion of an F (ab') 2 fragment would include DNA sequences encoding the CH 1 domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.
  • the murine, human or murine and chimeric antibodies, fragments and regions of the present invention are produced by cloning DNA segments encoding the H and L chain antigen-binding regions of a TNF-specific antibody, and joining these DNA segments to DNA segments encoding C H and C L regions, respectively, to produce murine, human or chimeric immunoglobulin-encoding genes.
  • a fused chimeric gene which comprises a first DNA segment that encodes at least the antigen-binding region of non-human origin, such as a functionally rearranged V region with joining (J) segment, linked to a second DNA segment encoding at least a part of a human C region.
  • cDNA encoding the antibody V and C regions the method of producing the chimeric antibody according to the present invention involves several steps, outlined below:
  • H and L chain J regions have different sequences, but a high degree of sequence homology exists (greater than 80%) among each group, especially near the C region. This homology is exploited in this method and consensus sequences of H and L chain J regions may be used to design oligonucleotides for use as primers for introducing useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments.
  • C region cDNA vectors prepared from human cells can be modified by site-directed mutagenesis to place a restriction site at the analogous position in the human sequence. For example, one can clone the complete human kappa chain C (C k ) region and the complete human gamma-1 C region (C gamma-1 ). In this case, the alternative method based upon genomic C region clones as the source for C region vectors would not allow these genes to be expressed in bacterial systems where enzymes needed to remove intervening sequences are absent. Cloned V region segments are excised and ligated to L or B chain C region vectors.
  • the human C gamma-1 region can be modified by introducing a termination codon thereby generating a gene sequence which encodes the H chain portion of an Fab molecule.
  • the coding sequences with linked V and C regions are then transferred into appropriate expression vehicles for expression in appropriate hosts, prokaryotic or eukaryotic.
  • Two coding DNA sequences are said to be "operably linked” if the linkage results in a continuously translatable sequence without alteration or interruption of the triplet reading frame.
  • a DNA coding sequence is operably linked to a gene expression element if the linkage results in the proper function of that gene expression element to result in expression of the coding sequence.
  • a chimeric antibody such as a mouse-human or human-human, will typically be synthesized from genes driven by the chromosomal gene promoters native to the mouse H and L chain V regions used in the constructs; splicing usually occurs between the splice donor site in the mouse J region and the splice acceptor site preceding the human C region and also at the splice regions that occur within the human C H region; polyadenylation and transcription termination occur at native chromosomal sites downstream of the human coding regions.
  • Gene expression elements useful for the expression of cDNA genes include: (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter ( Okayama, H. et al., Mol. Cell. Biol. 3:280 (1983 )), Rous sarcoma virus LTR ( Gorman, C. et al. , Proc, Natl. Acad, Sci., USA 79:6777 (1982 )), and Moloney murine leukemia virus LTR ( Grosschedl, R. et al., cell 41:885 (1985 )); (b) splice regions and polyadenylation sites such as those derived from the SV40 late region (Okayama et al. , supra ); and (c) polyadenylation sites such as in SV40 (Okayama et al. , supra ).
  • viral transcription promoters and their enhancer elements such as the SV40 early promoter ( Okayama, H. et al., Mol.
  • the transcriptional promoter is human cytomegalovirus
  • the promoter enhancers are cytomegalovirus and mouse/human immunoglobulin
  • mRNA splicing and polyadenylation regions are from the native chromosomal immunoglobulin sequences.
  • Each fused gene is assembled in, or inserted into, an expression vector.
  • Recipient cells capable of expressing the chimeric immunoglobulin chain gene product are then transfected singly with a chimeric H or chimeric L chain-encoding gene, or are co-transfected with a chimeric H and a chimeric L chain gene.
  • the transfected recipient cells are cultured under conditions that permit expression of the incorporated genes and the expressed immunoglobulin chains or intact antibodies or fragments are recovered from the culture.
  • the fused genes encoding the chimeric H and L chains, or portions thereof are assembled in separate expression vectors that are then used to co-transfect a recipient cell.
  • Each vector may contain two selectable genes, a first selectable gene designed for selection in a bacterial system and a second selectable gene designed for selection in a eukaryotic system, wherein each vector has a different pair of genes.
  • This strategy results in vectors which first direct the production, and permit amplification, of the fused genes in a bacterial system.
  • the genes so produced and amplified in a bacterial host are subsequently used to co-transfect a eukaryotic cell, and allow selection of a co-transfected cell carrying the desired transfected genes.
  • selectable genes for use in a bacterial system are the gene that confers resistance to ampicillin and the gene that confers resistance to chloramphenicol.
  • Preferred selectable genes for use in eukaryotic transfectants include the xanthine guanine phosphoribosyl transferase gene (designated gpt ) and the phosphotransferase gene from Tn5 (designated neo ). Selection of cells expressing gpt is based on the fact that the enzyme encoded by this gene utilizes xanthine as a substrate for purine nucleotide synthesis, whereas the analogous endogenous enzyme cannot.
  • the two selection procedures can be used simultaneously or sequentially to select for the expression of immunoglobulin chain genes introduced on two different DNA vectors into a eukaryotic cell. It is not necessary to include different selectable markers for eukaryotic cells; an H and an L chain vector, each containing the same selectable marker can be co-transfected. After selection of the appropriately resistant cells, the majority of the clones will contain integrated copies of both H and L chain vectors.
  • the fused genes encoding the chimeric H and L chains can be assembled on the same expression vector.
  • the preferred recipient cell line is a myeloma cell.
  • Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded by transfected immunoglobulin genes and possess the mechanism for glycosylation of the immunoglobulin.
  • a particularly preferred recipient cell is the Ig-non-producing myeloma cell SP2/0 (ATCC #CRL 8287). SP2/0 cells produce only immunoglobulin encoded by the transfected genes.
  • Myeloma cells can be grown in culture or in the peritoneal cavity of a mouse, where secreted immunoglobulin can be obtained from ascites fluid.
  • Other suitable recipient cells include lymphoid cells such as B lymphocytes of human or non-human origin, hybridoma cells of human or non-human origin, or interspecies heterohybridoma cells.
  • the expression vector carrying a chimeric antibody construct of the present invention may be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile bombardment ( Johnston et al., Science 240:1538 (1988 )).
  • a preferred way of introducing DNA into lymphoid cells is by electroporation ( Potter et al., Proc. Natl. Acad. Sci. USA 81:7161 (1984 ) ; Yoshikawa, K.
  • Yeast provides substantial advantages over bacteria for the production of immunoglobulin H and L chains. Yeasts carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies now exist which utilize strong promoter sequences and high copy number plasmids which can be used for production of the desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides) ( Hitzman, et al., 11th International Conference on Yeast, Genetics and Molecular Biology, adjoin, France, September 13-17, 1982 ).
  • Yeast gene expression systems can be routinely evaluated for the levels of production, secretion and the stability of chimeric H and L chain proteins and assembled murine and chimeric antibodies, fragments and regions .
  • Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeasts are grown in media rich in glucose can be utilized.
  • Known glycolytic genes can also provide very efficient transcription control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized.
  • PGK phosphoglycerate kinase
  • Bacterial strains may also be utilized as hosts for the production of antibody molecules or antibody fragments described by this invention, E. coli K12 strains such as E, coli W3110 (ATCC 27325), and other enterobacteria such as Salmonella typhimurium or Serratia marcescens , and various Pseudomonas species may be used.
  • E. coli K12 strains such as E, coli W3110 (ATCC 27325)
  • enterobacteria such as Salmonella typhimurium or Serratia marcescens
  • various Pseudomonas species may be used.
  • Plasmid vectors containing replicon and control sequences which are derived from species compatible with a host cell are used in connection with these bacterial hosts.
  • the vector carries a replication site, as well as specific genes which are capable of providing phenotypic selection in transformed cells.
  • a number of approaches may be taken for evaluating the expression plasmids for the production of murine and chimeric antibodies, fragments and regions or antibody chains encoded by the cloned immunoglobulin cDNAs in bacteria (see Glover, D.M., ed., DNA Cloning, Vol. I, IRL Press, 1985 ).
  • Preferred hosts are mammalian cells, grown in vitro or in vivo .
  • Mammalian cells provide post-translational modifications to immunoglobulin protein molecules including leader peptide removal, folding and assembly of H and L chains, glycosylation of the antibody molecules, and secretion of functional antibody protein.
  • Mammalian cells which may be useful as hosts for the production of antibody proteins include cells of fibroblast origin, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61).
  • H and L chain genes are available for the expression of cloned H and L chain genes in mammalian cells (see Glover, D.M., ed., DNA Cloning, Vol. II, pp141-238, IRL Press, 1985 ). Different approaches can be followed to obtain complete H 2 L 2 antibodies. As discussed above, it is possible to co-express H and L chains in the same cells to achieve intracellular association and linkage of H and L chains into complete tetrameric H 2 L 2 antibodies. The co-expression can occur by using either the same or different plasmids in the same host. Genes for both H and L chains can be placed into the same plasmid, which is then transfected into cells, thereby selecting directly for cells that express both chains.
  • cells may be transfected first with a plasmid encoding one chain, for example the L chain, followed by transfection of the resulting cell line with an H chain plasmid containing a second selectable marker.
  • Cell lines producing H 2 L 2 molecules via either route could be transfected with plasmids encoding additional copies of H, L, or H plus L chains in conjunction with additional selectable markers to generate cell lines with enhanced properties, such as higher production of assembled H 2 L 2 antibody molecules or enhanced stability of the transfected cell lines.
  • an anti-idiotypic (anti-Id) antibody specific for the anti-TNF antibody of the invention is an antibody which recognizes unique determinants generally associated with the antigen-binding region of another antibody.
  • the antibody specific for TNF is termed the idiotypic or Id antibody.
  • the ariti-Id can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the Id antibody with the Id antibody or the antigen-binding region thereof. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an anti-Id antibody.
  • the anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • the anti-anti-Id may be epitopically identical to the original antibody which induced the anti-Id.
  • antibodies to the idiotypic determinants of a mAb it is possible to identify other clones expressing antibodies of identical specificity.
  • mAbs generated against TNF may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice.
  • Spleen cells from such immunized mice can be used to produce anti-Id hybridomas secreting anti-Id mAbs.
  • the anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional BALB/c mice.
  • KLH keyhole limpet hemocyanin
  • Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for a TNF epitope.
  • the antibodies, fragments and derivatives of the present invention are useful for treating a subject having a pathology or condition associated with levels of a substance reactive with an anti-TNF antibody, in particular TNF, in excess of the levels present in a normal healthy subject.
  • pathologies include, but are not limited to, sepsis syndrome, including cachexia, circulatory collapse and shock resulting from acute or chronic bacterial infection, acute and chronic parasitic or infectious processes, including bacterial, viral and fungal infections, acute and chronic immune and autoimmune pathologies, such as systemic lupus erythematosus and rheumatoid arthritis, alcohol-induced hepatitis, chronic inflammatory pathologies such as sarcoidosis and Crohn's pathology, vascular inflammatory pathologies such as disseminated intravascular coagulation, graft-versus-host pathology, Kawasaki's pathology and malignant pathologies involving TNF-secreting tumors.
  • Such treatment comprises parenterally administering a single or multiple doses of the antibody, fragment or derivative.
  • Preferred for human pharmaceutical use are high affinity potent hTNF ⁇ -inhibiting and/or neutralizing murine and chimeric antibodies, fragments and regions of this invention.
  • Monoclonal antibodies may be administered by any means that enables the active agent to reach the agent's site of action in the body of a mammal.
  • the primary focus is the ability to reach and bind with TNF released by monocytes and macrophages. Because proteins are subject to being digested when administered orally, parenteral administration, i.e., intravenous, subcutaneous, intramuscular, would ordinarily be used to optimize absorption.
  • Monoclonal antibodies may be administered either as individual therapeutic agents or in combination with other therapeutic agents. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • the dosage administered will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
  • a daily dosage of active ingredient can be about 0.1 to 100 milligrams per kilogram of body weight.
  • 0.5 to 50, and preferably 1 to 10 milligrams per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.
  • Dosage forms (composition) suitable for internal administration generally contain from about 1 milligram to about 500 milligrams of active ingredient per unit.
  • the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition.
  • the antibody can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle examples include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used.
  • the vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques.
  • Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.
  • a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution.
  • the antibodies of this invention can be adapted for therapeutic efficacy by virtue of their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells having TNF associated with their surface.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • an endogenous source or an exogenous source of effector cells (for ADCC) or complement components (for CDC) can be utilized.
  • the murine and chimeric antibodies, fragments and regions of this invention, their fragments, and derivatives can be used therapeutically as immunoconjugates (see for review: Dillman, R.O., Ann. Int. Med. 111:592-603 (1989 )).
  • cytotoxic proteins including, but not limited to Ricin-A, Pseudomonas toxin, Diphtheria toxin, and TNF.
  • Toxins conjugated to antibodies or other ligands are known in the art (see, for example, Olsnes. S. et al., Immunol. Today 10:291-295 (1989 )). Plant and bacterial toxins typically kill cells by disrupting the protein synthetic machinery.
  • the antibodies of this invention can be conjugated to additional types of therapeutic moieties including, but not limited to, radionuclides, cytotoxic agents and drugs.
  • radionuclides which can be coupled to antibodies and delivered in vivo to sites of antigen include 212 Bi, 131 I, 186 Re, and 90 Y, which list is not intended to be exhaustive.
  • the radionuclides exert their cytotoxic effect by locally irradiating the cells, leading to various intracellular lesions, as is known in the art of radiotherapy.
  • the antibodies of this invention may be advantageously utilized in combination with other monoclonal or murine and chimeric antibodies, fragments and regions, or with lymphokines or hemopoietic growth factors, etc., which serve to increase the number or activity of effector cells which interact with the antibodies.
  • the antibodies, fragments or derivatives of this invention may also be used in combination with TNF therapy to block undesired side effects of TNF.
  • Recent approaches to cancer therapy have included direct administration of TNF to cancer patients or immunotherapy of cancer patients with lymphokine activated killer (LAK) cells ( Rosenberg et al., New Eng. J. Med. 313:1485-1492 (1985 )) or tumor infiltrating lymphocytes (TIL) ( Kurnick et al. (Clin. Immunol. Immunogath, 38:367-380 (1986 ) ; Kradin et al., Cancer Immunol. Immunother, 24:76-85 (1987 ); Kradin et al., Transplant, Proc, 20:336-338 (1988) ).
  • LAK lymphokine activated killer
  • TIL tumor infiltrating lymphocytes
  • the murine and chimeric antibodies, fragments and regions, fragments, or derivatives of this invention, attached to a solid support can be used to remove TNF from fluids or tissue or cell extracts. In a preferred embodiment, they are used to remove TNF from blood or blood plasma products. In another preferred embodiment, the murine and chimeric antibodies, fragments and regions are advantageously used in extracorporeal immunoadsorbent devices, which are known in the art (see, for example, Seminars in Hematology, Vol. 26 (2 Suppl. 1). (1989 )). Patient blood or other body fluid is exposed to the attached antibody, resulting in partial or complete removal of circulating TNF (free or in immune complexes), following which the fluid is returned to the body.
  • This immunoadsorption can be implemented in a continuous flow arrangement, with or without interposing a cell centrifugation step. See, for example, Terman, D.S. et al., J. Immunol. 117:1971-1975 (1976 ).
  • the present invention also provides the above antibodies, fragments and derivatives, detectably labeled, as described below, for use in diagnostic methods for detecting TNF ⁇ in patients known to be or suspected of having a TNF ⁇ -mediated condition.
  • the antibodies of the present invention are useful for immunoassays which detect or quantitate TNF, or anti-TNF antibodies, in a sample.
  • An immunoassay for TNF typically comprises incubating a biological sample in the presence of a detectably labeled high affinity antibody of the present invention capable of selectively binding to TNF, and detecting the labeled antibody which is bound in a sample.
  • Various clinical immunoassay procedures are described in Immunoassays for the 80's, A. Voller et al., eds., University Park, 1981 .
  • solid phase support or “carrier” is intended any support capable of binding antigen or antibodies.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to TNF or an anti-TNF antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • TNF-specific antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA), or enzyme-linked immunosorbent assay (ELISA).
  • EIA enzyme immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • Enzymes which can be used to detectably label the TNF-specific antibodies of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • radioactively labeling the TNF-specific antibodies By radioactively labeling the TNF-specific antibodies, it is possible to detect TNF through the use of a radioimmunoassay (RIA) (see, for example, Work, T.S., et al., Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, N.Y. (1978 ).
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • Isotopes which are particularly useful for the purpose of the present invention are: 3 H, 125 I, 131 I, 35 S, 14 C, and, preferably, 125 I.
  • TNF-specific antibodies it is also possible to label the TNF-specific antibodies with a fluorescent compound.
  • fluorescent labeled antibody When the fluorescent labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence.
  • fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o -phthaldehyde and fluorescamine.
  • the TNF-specific antibodies can also be detectably labeled using fluorescence-emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the TNF-specific antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediamine-tetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediamine-tetraacetic acid
  • the TNF-specific antibodies also can be detectably labeled by coupling to a chemiluminescent compound.
  • the presence of the chemiluminescently labeled antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the TNF-specific antibody, fragment or derivative of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • Detection of the TNF-specific antibody, fragment or derivative may be accomplished by a scintillation counter, for example, if the detectable label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorescent material.
  • the detection can be accomplished by colorometric methods which employ a substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • the TNF which is detected by the above assays may be present in a biological sample.
  • a biological sample such as, for example, blood, serum, lymph, urine, inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissue extract or homogenate, and the like.
  • the invention is not limited to assays using only these samples, it being possible for one of ordinary skill in the art to determine suitable conditions which allow the use of other samples.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and providing the combination of labeled antibodies of the present invention to such a specimen.
  • the antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample.
  • the antibody, fragment or derivative of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site” or “sandwich” assay.
  • an immunometric assay also known as a "two-site” or “sandwich” assay.
  • a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support that is insoluble in the fluid being tested and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
  • Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the TNF from the sample by formation of a binary solid phase antibody-TNF complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted TNF, if any, and then contacted with the solution containing a known quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the TNF bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody.
  • This type of forward sandwich assay may be a simple "yes/no” assay to determine whether TNF is present or may be made quantitative by comparing the measure of labeled antibody with that obtained for a standard sample containing known quantities of TNF.
  • Such "two-site” or “sandwich” assays are described by Wide (Radioimmune Assay, Method, Kirkham, ed., E. & S. Livingstone, Edinburgh, 1970, pp. 199-206 ).
  • a simultaneous assay involves a single incubation step wherein the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is' washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
  • stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period, is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward” assays.
  • a combination of antibodies of the present invention specific for separate epitopes may be used to construct a sensitive three-site immunoradiometric assay.
  • Spleen cells were fused with cells of the nonsecreting hybridoma, Sp2/0 (ATCC CRL1581), at a 4:1 ratio of spleen cells to Sp2/0 cells, in the presence of 0.3 ml of 30% polyethylene glycol, PEG 1450. After incubation at 37°C for 6 hours, the fused cells were distributed in 0.2 ml aliquots into 96-well plates at concentrations of 2 x 10 4 SP2/0 cells per well. Feeder cells, in the form of 5 x 10 4 normal BALB/c spleen cells, were added to each well.
  • the growth medium used consisted of RPM1-1640 medium, 10% heat-inactivated fetal bovine serum (FBS) (Hyclone), 0.1 mM MEM nonessential amino acids, 1 mM sodium pyruvate, 2mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin (GIBCO Laboratories) and, for selection, hypoxanthine-aminopterin-thymidine (HAT) (Boehringer Mannheim).
  • FBS heat-inactivated fetal bovine serum
  • HAT hypoxanthine-aminopterin-thymidine
  • a solid-phase radioimmunoassay was employed for screening supernatants for the presence of mAbs specific for rhTNF ⁇ . This assay is described in Example II, below. The background binding in this assay was about 500 cpm. A supernatant was considered positive if it yielded binding of 2000 cpm or higher.
  • the hybridoma line A2 was selected. This line was maintained in RPM1-1640 medium with 10% FBS (GIBCO), 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
  • anti-TNF antibodies which inhibit TNF biological activity can be screened by binding to peptide including at least 5 amino acids of residues 87-108 or both residues 59-80 and 87-108 of TNF (of SEQ ID NO:1) or combinations of peptides contained therein, which are used in place of the rTNF protein, as described above.
  • E. coli -derived rhTNF was diluted to 1 ⁇ g/ml in BCB buffer, pH 9.6, and 0.1 ml of the solution was added to each assay well. After incubation at 4°C overnight, the wells were washed briefly with BCB, then sealed with 1% bovine serum albumin (BSA) in BCB at 37°C for 1 hr. The wells were then washed 3 times with PBS containing 0.05% Tween-20 (PBS-Tween), and 70 ⁇ l diluted A2 ascites fluid was added to each well. The wells were incubated for 2 hr at 37°C, and washed 3 times with PBS-Tween.
  • BSA bovine serum albumin
  • Samples of A2 and cA2 were purified by protein A affinity chromatography from hybridoma tissue culture supernatants of cell lines designated C134A and C168A (described above), respectively, and diafiltered in phosphate buffered saline pH 7.2 (PBS) .
  • PBS phosphate buffered saline pH 7.2
  • TNF-sensitive target cell A673 a human rhabdomyosarcoma cell line
  • A673/6 cells were seeded at 3 x 10 4 cells/well 20 hr before the TNF bioassay.
  • Two-fold serial dilutions of rhTNF, E. coli -derived recombinant human lymphotoxin (TNF ⁇ ), and E. coli -derived recombinant murine TNF were prepared.
  • the A2 hybridoma supernatant was added to an equal volume of the diluted TNF preparations, and the mixtures were incubated at room temperature for 30 min.
  • TNF-1, TNF-2 and TNF-3 The inhibiting and/or neutralizing activity of mAb A2 was compared with three other murine mAbs specific for human TNF, termed TNF-1, TNF-2 and TNF-3, and a control mAb.
  • TNF-1, TNF-2 and TNF-3 Two-fold serial dilutions of purified mAbs were mixed with rhTNF (40 pg/ml), incubated at room temperature for 30 min, and aliquots tested for TNF bioactivity as above. It was found that mAbs TNF-1, TNF-2 and TNF-3 each had a similar moderate degree of inhibiting and/or neutralizing activity. In contrast, mAb A2 had much more potent inhibiting and/or neutralizing activity.
  • J region DNA probes can be used to screen genomic libraries to isolate DNA linked to the J regions. Although DNA in the germline configuration (i.e., unrearranged) would also hybridize to J probes, this DNA would not be linked to a Ig V region sequence and can be identified by restriction enzyme analysis of the isolated clones.
  • the cloning utilized herein was to isolate V regions from rearranged H and L chain genes using J H and J k probes. These clones were tested to see if their sequences were expressed in the A2 hybridoma by Northern analysis. Those clones that contained expressed sequence were cloned into expression vectors containing human C regions and transfected into mouse myeloma cells to determine if an antibody was produced. The antibody from producing cells was then tested for binding specificity and functionally compared to the A2 murine antibody.
  • DNA fragments from each size class were ligated with lambda charon 27 arms and packaged into phage particles in vitro using Gigapack Gold from Stratagene (LaJolla, CA).
  • the mouse L chain J k probe was a 2.7 kb Hind III fragment containing all five J k segments.
  • the probe was labeled with 32 P by random priming using a kit obtained from Boehringer Mannheim. Free nucleotides were removed by centrifugation through a Sephadex G-50 column. The specific activities of the probe was approximately 10 9 cpm/ ⁇ g.
  • Plaque hybridizations were carried out in 5x SSC, 50% formamide, 2x Denhardt's reagent, and 200 ⁇ g/ml denatured salmon sperm DNA at 42°C for 18-20 hours. Final washes were in 0.5x SSC, 0.1% SDS at 65°C. Positive clones were identified after autoradiography.
  • V region gene for the A2 H chain was isolated in the lambda gt10 vector system. High molecular weight DNA was digested to completion with restriction endonuclease EcoR I and fragments of approximately 7.5 kb were isolated after agarose gel electrophoresis. These fragments were ligated with lambda gt10 arms and packaged into phage particles in vitro using Gigapack Gold.
  • This library was screened at a density of 20,000 plaques per 150 mm plate using a J H probe.
  • the J H probe was a 2kb BamH I/ EcoR I fragment containing both J3 and J4 segments.
  • the probe was labeled as in Example III and had a similar specific radioactivity. Hybridization and wash conditions were identical to those used in Example III.
  • the observed lengths of hybridizing A2 mRNA were the correct sizes for H and L chain mRNA, respectively. Because the RNA expression was restricted to the A2 hybridoma, it was assumed that the 7.5 kb H chain fragments and the 2.9 kb L chain fragments contained the correct V region sequences from A2. One example of each type was chosen for further study. The important functional test is the demonstration that these V regions sequences, when combined with appropriate C region sequences, are capable of directing the synthesis of an antibody with a specificity and affinity similar to that of the murine A2 antibody.
  • the 7.5 kb H chain fragment and the 2.9 kb L chain fragment were subcloned into plasmid vectors that allow expression of the chimeric mouse/human proteins in murine myeloma cells (see Examples VIII and IX). These plasmids were co-transfected into SP2/0 cells to ascertain if intact antibody molecules were secreted, and if so, if they were of the correct specificity and affinity. Control transfections were also performed pairing the putative anti-TNF H chain with an irrelevant, but expressed, L chain; the putative anti-TNF L chain was also paired with an irrelevant, but expressed, H chain. The results indicated that the 7.5 kb H chain fragment could be expressed, whereas the 2.9 kb L chain fragment could not. This was confirmed by DNA sequence analysis that suggested portions of the coding region were not in the proper amino acid reading frame when compared to other known L chain amino acid sequences.
  • the 4.0 kb and 5.7 kb Hind III fragments isolated from L chain libraries were cloned into expression vectors and tested for expression of chimeric antibody after co-transfeation with the 7.5 kb H chain.
  • the 5.7 kb Hind III fragment was incapable of supporting antibody expression, whereas the 4.0 kb Hind III fragment did support antibody expression.
  • the antibody resulting from the co-transfection of the 7.5 kb putative H chain V region and the 4.0 kb L chain V region was purified, tested in solid phase TNF binding assay, and found to be inactive.
  • the 2.9 kb Hind III fragment from clone 4.3 was subcloned into the L chain expression vector and co-transfected with the putative anti-TNF H chain into SP2/0 cells. An antibody was synthesized, purified and tested in the solid phase TNF binding assay. This antibody bound to TNF, and therefore, the clone 4.3 L chain V region was assumed to be the correct one.
  • the A2 murine hybridoma has been shown to contain at least four rearranged L chain V region genes. At least two of these are expressed as proteins: clone 4.3 (the correct anti-TNF L chain gene) and the gene contained in the 4.0 kb Hind III fragment (contributed by the fusion partner).
  • the expression of two L chains implies that the resulting antibody secreted from the murine hybridoma is actually a mixture of antibodies, some using the correct L chain, some using the incorrect L chain, and some using one of each.
  • the presence of two different L chains in the murine A2 antibody has been confirmed by SDS gel and N-terminal protein sequence analysis of the purified antibody.
  • the resulting antibody will have only the correct L chain and therefore should be a more potent antibody (see Examples X, XI and XII).
  • RNA was subjected to electrophoresis on 1% agarose/formaldehyde gels (Sambrook et al , supra ) and transferred to nitrocellulose. Blots were hybridized with random primed DNA probes in 50% formamide, 2x Denhardt's solution, 5x SSC, and 200 ⁇ g/ml denatured salmon sperm DNA at 42°C for 10 hours. Final wash conditions were 0.5 x SSC, 0.1% SDS at 65°C.
  • the subcloned DNA fragments were labeled with 32 P by random priming and hybridized to Northern blots containing total RNA derived from A2 cells or from cells of SP2/0, the fusion partner parent of A2.
  • the 7.5 kb EcoR I H chain fragment hybridized with a 2 kb mRNA from A2, but not with SP2/0 mRNA.
  • the 2.9 kb L chain Hind III fragment hybridized with a 1250 bp mRNA from A2, but not with SP2/0 mRNA.
  • the observed lengths of A2 mRNA hybridizing were the correct sizes for H and L chain mRNA, respectively, confirming that the V region sequences on these DNA fragments are expressed in A2 hybridoma cells.
  • the putative L (clone 4.3) and H chain V genes described above were joined to human kappa and gamma1 constant region genes in expression vectors.
  • the 7.5 kb EcoR I fragment corresponding to the putative V H region gene from A2 was cloned into an expression vector containing the human C gamma1 gene and the Ecogpt gene to yield the plasmid designated pA2HG1apgpt (see Figure 8).
  • the 2.9 kb putative V L fragment from clone 4.3 was cloned into a vector containing the human kappa C k gene and the Ecogpt gene to allow selection in mammalian cells.
  • the resulting plasmid was designated pA2HuKapgpt (See Figure 8).
  • Plasmid DNA to be transfected was purified by centrifuging to equilibrium in ethidium bromide/cesium chloride gradients twice. Plasmid DNA (10-50 ⁇ g) was added to 10 7 SP2/0 cells in medium containing Hank's salts, and the mixture was placed in a BioRad electroporation apparatus. Electroporation was performed at 20 volts, following which the cells were plated in 96 well microtiter plates.
  • the chimeric A2 antibody was purified from tissue culture supernatant by Protein A- Sepharose chromatography. The supernatant was adjusted to 0.1M Tris, 0.002M EDTA, pH 8.0 and loaded on a Protein A-Sepharose column equilibrated in the same buffer. The IgG was eluted with 0.1M citrate, pH 3.5, inhibited or neutralized with 1M Tris, and dialyzed into phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the purified chimeric antibody was evaluated for its binding and inhibiting and/or neutralizing activity.
  • the affinity constant for binding of mouse mAb A2 and cA2 to rhTNF ⁇ was determined by Scatchard analysis (see, for example, Scatchard, G., Ann. N.Y. Acad. Sei. 51:660 (1949 )). The results are shown in Figure 10. This analysis involved measuring the direct binding of 125 I labelled cA2 to immobilized rhTNF ⁇ in a 96-well plate. The antibodies were each labelled to a specific activity of about 9.7 ⁇ Ci/ ⁇ g by the iodogen method. An affinity constant (Ka) of 0.5 x 10 9 liters/mole was calculated for the mouse mAb A2.
  • the chimeric A2 antibody had a higher affinity, with a Ka of 1.8 x 10 9 liters/mole.
  • the chimeric anti-TNF ⁇ antibody of the present invention was shown to exhibit a significantly higher affinity of binding to human TNF ⁇ than did the parental murine A2 mAb. This finding was surprising, since murine and chimeric antibodies, fragments and regions would be expected to have affinities that are equal to or less than that of the parent mAb.
  • Such high affinity anti-TNF antibodies having affinities of binding to TNF ⁇ of at least 1 x 10 8 M -1 , more preferably at least 1 x 10 9 M -1 (expressed as Ka) are preferred for immunoassays which detect very low levels of TNF in biological fluids.
  • anti-TNF antibodies having such high affinities are preferred for therapy of TNF- ⁇ -mediated conditions or pathology states.
  • cA2 for TNF was confirmed by testing for cross-neutralization of human lymphotoxin (TNF- ⁇ ). Lymphotoxin shares some sequence homology and certain biological activities, for example, tumor cell cytotoxicity, with TNF ( Pennica, D. et al., Nature 312:724-729 (1984 )). Cultured human A673 cells were incubated with increasing concentrations of human lymphotoxin (Genentech, San Francisco, CA) with or without 4 ⁇ g/ml chimeric A2 in the presence of 20 ⁇ g/ml cycloheximide at 39°C overnight. Cell death was measured by vital staining with naphthol blue-black, as above. The results indicated that cA2 was ineffective at inhibiting and/or neutralizing human lymphotoxin, confirming the TNF ⁇ -specificity of the chimeric antibody.
  • A2 or cA2 The ability of A2 or cA2 to react with TNF from different animal species was also evaluated.
  • TNF TNF has bioactivity in a wide range of host animal species.
  • certain inhibiting and/or neutralizing epitopes on human TNF are conserved amongst different animal species and others appear to be restricted to humans and chimpanzees.
  • Neutralization experiments utilized endotoxin-activated cell supernatants from freshly isolated human, chimpanzee, rhesus and cynomolgus monkey, baboon, pig, dog, rabbit, or rat monocytes as the TNF source.
  • murine mAb A2 inhibited or neutralized activity of only human and chimpanzee TNF, and had no effect on TNF derived from other primates and lower animals. A2 also did not inhibit or neutralize the cytotoxic effect of recombinant mouse TNF.
  • the epitope recognized by A2 is one shared by human and chimpanzee TNF ⁇ .
  • Chimeric A2 was also tested in this manner for cross-reactivity with monocyte-derived TNF from rat, rabbit, dog and pig, as well as with purified recombinant mouse TNF ⁇ , and natural and recombinant human TNF ⁇ .
  • Chimeric A2 only inhibited or neutralized natural and recombinant human TNF ⁇ . Therefore, cA2 appears to share species specificity with murine A2.
  • Both the murine and chimeric anti-TNF ⁇ antibodies, A2 and cA2 were determined to have potent TNF-inhibiting and/or neutralizing activity.
  • murine A2 at a concentration of about 125 ng/ml completely inhibited or neutralized the biological activity of a 40 pg/ml challenge of rhTNF ⁇ .
  • Two separate determinations of inhibiting and/or neutralizing potency, expressed as the 50% Inhibitory Dose (ID50) were determined to be 15.9 ⁇ 1.01 and 17.9 ⁇ 1.6 ng/ml (Mean ⁇ Std error).
  • ID50 50% Inhibitory Dose
  • TNF-1 murine anti-TNF ⁇ antibodies
  • TNF-2 murine anti-TNF ⁇ antibodies
  • cA2 The ability of cA2 to inhibit or neutralize human TNF ⁇ bioactivity in vitro was tested using the bioassay system described above. Cultured A673 cells were incubated with 40 pg/ml natural (Genzyme, Boston, MA) or recombinant (Suntory, Osaka, Japan) human TNF with or without antibody overnight as above, and cell death was measured by vital staining. As expected based upon the above results with the A2 mouse mAb, cA2 also inhibited or neutralized both natural and rhTNF in a dose-dependent manner in the cytotoxicity assay (Figure 11). In this assay format, levels of cA2 as low as 125 ng/ml completely abolished the toxic activity of TNF.
  • the cA2 Upon repeated analysis, the cA2 exhibited greater TNF-inhibiting and/or neutralizing activity than did the parent murine A2 mAb.
  • Such inhibiting and/or neutralizing potency at antibody levels below 1 ⁇ g/ml, can easily be attained in the blood of a subject to whom the antibody is administered. Accordingly, such highly potent inhibiting and/or neutralizing anti-TNF antibodies, in particular the chimeric antibody, are preferred for therapeutic use in TNF ⁇ -mediated pathologies or conditions.
  • TNF induces cellular secretion of IL-6.
  • IL-6 is involved in the pathophysiology of sepsis, although the precise role of IL-6 in that syndrome is unclear ( Fong, Y. et al., J Exp Med 170:1627-1633 (1989 ); Starnes Jr., H.F. et al., J Immunol 145:4185-4191 (1990 )).
  • the ability of cA2 to inhibit or neutralize TNF-induced IL-6 secretion was evaluated using cultured human diploid FS-4 fibroblasts. The results in Table 1 show that cA2 was effective in blocking IL-6 secretion in cells that had been incubated overnight with TNF.
  • TNF-induced IL-6 secretion was not inhibited in the absence of a mAb or in the presence of a control mAb specific for an irrelevant antigen.
  • TNF Concentration (ng/ml) Antibody 0 0.3 1.5 7.5 None ⁇ 0.20 1.36 2.00 2_56
  • Control mAb ⁇ 0.20 1.60 1.96
  • cA2 ⁇ 0.20 ⁇ 0.20 ⁇ 0.20 0.30
  • Values represent mean concentrations of IL-6 of duplicate wells, in ng/ml.
  • RhTNF (Suntory, Osaka, Japan), with or without 4 ⁇ g/ml antibody, was added to cultures of FS-4 fibroblasts and after 18 h, the supernatant was assayed for IL-6 using the Quantikine Human IL-6 Immunoassay (from R&D Systems, Minneapolis, MN).
  • Control mAb chimeric mouse/human IgG1 anti-platelet mAb (7E3).
  • TNF procoagulant and adhesion molecule activities of endothelial cells
  • EC endothelial cells
  • TNF stimulation of procoagulant activity was determined by exposing intact cultured HDVEC cells to TNF (with or without antibody) for 4 hours and analyzing a cell lysate in a human plasma clotting assay.
  • TNF In addition to stimulating procoagulant activity, TNF also induces surface expression of endothelial cell adhesion molecules such as ELAM-1 and ICAM-1.
  • endothelial cell adhesion molecules such as ELAM-1 and ICAM-1.
  • the ability of cA2 to inhibit or neutralize this activity of TNF was measured using an ELAM-1 specific detection radioimmunoassay.
  • Cultured HUVEC were stimulated with 250 ng/ml rhTNF (Dainippon, Osaka, Japan) with or without antibody at 37°C overnight in a 96-well plate format.
  • Surface expression of ELAM-1 was determined by sequential addition of a mouse anti-human ELAM-1 mAb and 125 I-labelled rabbit anti-mouse immunoglobulin second antibody directly to culture plates at 4°C.
  • TNF induced the expression of ELAM-1 on the surface of cultured HUVEC cells, and this activity was again effectively blocked in a dose-related manner by cA2.
  • TNF is known to stimulate mitogenic activity in cultured fibroblasts.
  • Chimeric A2 inhibited or neutralized TNF-induced mitogenesis of human diploid FS-4 fibroblasts cultures, confirming the potent inhibiting and/or neutralizing capability of cA2 against a broad spectrum of in vitro TNF biological activities.
  • Female C3H/HeN mice were administered 5 ⁇ g rhTNF (Dainippon, Osaka, Japan) + 18 mg galactosamine i.p. and antibody was administered 15-30 minutes later i.v. Deaths were recorded 48 h post-challenge.
  • FMOC 9-fluorenylmethoxycarbonyl
  • tBu t-butyl ether OtB, t-butyl ester
  • Boc t-butyloxycarbonyl
  • Mtr 4-methoxy-2,3,6-trimethylbenzenesulfonyl
  • Trt trityl
  • OPfp pentafluorophenylester
  • ODhbt oxo-benzotriazone ster
  • a chimeric antibody of the present invention designated cA2
  • cA2 was used to determine which portions of the TNF amino acid sequence were involved in inhibitory binding by the antibody by epitope mapping, whereby the amino acid sequences of TNF- ⁇ recognized by cA2 have been identified.
  • FIG. 14A shows the results of binding to the overlapping decapeptides that comprise the entire sequence of human TNF ⁇ .
  • the O.D. optional density correlates directly with the increased degree of cA2 binding.
  • Figure 14B shows the results of binding of cA2 to the same set of peptide pins in the presence of human TNF ⁇ . This competitive binding study delineates peptides which may show non-specific binding to cA2.
  • the mAb cA2 blocks the action of TNP- ⁇ without binding to the putative receptor binding locus, which can include one or more of, e.g., 11-13, 37-42, 49-57 or 155-157 of hTNF ⁇ (of SEQ ID NO:1).
  • Preferred anti-TNF mAbs are those that inhibit this binding of human TNF- ⁇ to its receptors by virtue of their ability to bind to one or more of these peptide sequences.
  • These antibodies can block the activity of TNF by virtue of binding to the cA2 epitope, such binding demonstrated to inhibit TNF activity.
  • the identification of those peptide sequences recognized by cA2 provides the information necessary to generate additional monoclonal antibodies with binding characteristics and therapeutic utility that parallel the embodiments of this application.
  • Peptide Pin Synthesis Using an epitope mapping kit purchased from Cambridge Research Biochemicals, Inc. (CRB), dodecapeptides corresponding to the entire sequence of human TNF- ⁇ were synthesized on polyethylene pins.
  • a synthesis schedule was generated using the CRB epitope mapping software. Prior to the first amino acid coupling, the pins were deprotected with a 20% piperidine in NMP solution for 30 minutes at room temperature. After deprotected, the pins were washed with NMP for five minutes at room temperature, followed by three methanol washes. Following the wash steps, the pins were allowed to air dry for at least 10 minutes.
  • the peptide pins were washed, deprotected and treated with 150 microliters of a solution containing NMP; acetic anhydride:triethylamine (5:2:1) for 90 minutes at 30°C, followed by the washing procedure outlined above.
  • the second set of peptide pins was deprotected by not acetylated to give free N-terminal amino groups.
  • the final deprotection of the peptides to remove the side chain protecting groups was done using a mixture of TFA:anisole:dithiothreitol, 95:2.5:2.5 (v/v/w) for four hours at ambient temperature. After deprotection, the pins were air dried for 10 minutes, followed by a 15 minute sonication in a solution of 0.1% HCl in methanol/distilled water (1:1). The pins dried over night and were then ready for testing.
  • Disruption Buffer Sodium dihydrogen phosphate (31.2 g, Sigma cat # S-0751 or equivalent) and sodium dodecylsulfate (20.0 g, Sigma cat # L-3771 or equivalent) were dissolved in 2.0 L of milliQ water. The pH was adjusted to 7.2 ⁇ 0.1 with 50% w/w sodium hydroxide (VWR cat # VW6730-3 or equivalent).
  • Blocking Buffer Sodium dihydrogen phosphate (0.39 g, Sigma cat #S-0751 or equivalent) disodium hydrogen phosphate (1.07 g, Baker cat # 3828-1 or equivalent) and sodium chloride (8.50 g, Baker cat # 3624-5 or equivalent were dissolved in 1.0 L of milliQ water. The pH was adjusted to 7.2 ⁇ 0.1 with 50% w/w sodium hydroxide (VWR cat VW6730-3 or equivalent) . Chicken egg albumin (10.0 g, Sigma cat #A-5503 or equivalent) and bovine serum albumin (10.0 g, Sigma, cat #A-3294 or equivalent) were dissolved at room temperature with gentle stirring. The solution was filtered, and to the solution was added Tween 20 (2.0 ml, Sigma cat #P-13.79 or equivalent). The solution was stirred gently at room temperature for 30 min, filtered and stored at 40°.
  • PBS/Tween 20 A 10 x concentrate was prepared by dissolving sodium dihydrogen phosphate (3.90 g, Sigma cat # S-0751 or equivalent), disodium hydrogen phosphate (10.70 g, Baker cat #3828-1 or equivalent) and sodium chloride (85.0 g, Baker cat #3624-5 or equivalent) in 1.0 L of milliQ water. The pH was adjusted to 7.2 ⁇ 0.1 with 50% w/w sodium hydroxide (VWR cat #VW 6730 or equivalent). To the solution was added Tween 20 (5.0 mL, Sigma cat #P-1379 or equivalent), and the mixture stirred gently. Just prior to use 100 mL of this solution was diluted to 1.0 L with milliQ water.
  • Substrate buffer was prepared by dissolving citric acid (4.20g, Malinckrodt cat #0627 or equivalent) and disodium hydrogen phosphate (7.10 g, Baker cat #3828-1 or equivalent) in 1.0 L of milliQ water. The pH was adjusted to 5.00 with 50% w/w sodium hydroxide (VWR cat #VW6730-3 or equivalent). Immediately prior to use an OPD substrate tablet (30 mg, Sigma cat #P-8412 or equivalent and 30% v/v hydrogen peroxide (40 ⁇ L, Sigma cat #P-1379 or equivalent) were added to the substrate buffer 25.0 mL). The solution was wrapped in foil and mixed thoroughly.
  • peptide pins Prior to use and after each subsequent use the peptide pins were cleaned using the following procedure. Disruption buffer (2.0 L) was heated to 60° and placed in an ultra-sonic bath in a fume hood. To the disruption buffer was added dithiolthreitol (2.5 g, Sigma cat #D-0632 or equivalent). The peptide pins were sonicated in this medium for 30 min, washed thoroughly with milliQ waster, suspended in a boiling ethanol bath for 2 min, and air-dried.
  • Disruption buffer 2.0 L
  • dithiolthreitol 2.5 g, Sigma cat #D-0632 or equivalent
  • Blocking buffer 200 ⁇ L was added to a 96 well disposable polystyrene Elisa plate and the peptide pins suspended in the wells. The peptide pins and plate were incubated for 2 h at room temperature on an oscillating table shaker. The plates and peptide pins were washed with PBS/Tween 20 (four times). To each well was added a 20 ⁇ g/ml concentration of cA2 antibody (diluted with blocking buffer, 175 ⁇ L/well). TNF competition was done by incubation of TNF ⁇ (40 ⁇ g/ml) and cA2 (20 ⁇ g/ml) in BSA/ovalbumin/BBS for three hours at room temperature.
  • the peptide pins were suspended in the plate and incubated at 4° overnight. The peptide pins and plate were washed with PBS/Tween 20 (four times). To each well was added anti-human goat antibody conjugated to horseradish peroxidase (diluted with blocking buffer to 1/2000, 175 ⁇ L/well, Jackson ImmunoResearch Labs). The peptide pins were suspended in the plate, and incubated for 1 h at room temperature on a oscillating table shaker. The plates and peptide pins were washed with PBS/Tween 20 (four times).
  • mice Female BALB/c mice, as in Example I above, are injected subcutaneously and intraperitoneally (i.p.) with forty ⁇ g of purified E. coli -derived recombinant human TNF (rhTNF) fragments comprising anti-TNF epitopes of at least 5 amino acids located within the non-contiguous sequence 59-80, 87-108 or both residues 59-80 and 87-108 of TNF (of SEQ ID NO:1), as presented above, emulsified with an equal volume of complete Freund's adjuvant (obtained from Difco Laboratories) in 0.4 ml is into a mouse.
  • rhTNF E. coli -derived recombinant human TNF
  • a booster injection of 5 ⁇ g of these rhTNF fragments in incomplete Freund's adjuvant is given i.p. followed by four consecutive i.p. injections of 10 ⁇ g of TNF fragments including anti-TNF epitopes including amino acids from residues 59-80, 87-108 or both 59-80 and 87-108 of hTNF ⁇ (of SEQ ID NO:1) without adjuvant.
  • the mouse is boosted i.p. with 10 ⁇ g of TNF.
  • Spleen cells are fused with cells of the nonsecreting hybridoma, Sp2/0 (ATCC CRL1581), at a 4:1 ratio of spleen cells to Sp2/0 cells, in the presence of 0.3 ml of 30% polyethylene glycol, PEG 1450. After incubation at 37°C for 6 hours, the fused cells are distributed in 0.2 ml aliquots into 96-well plates at concentrations of 2 x 10 4 SP2/0 cells per well. Feeder cells, in the form of 5 x 10 4 normal BALB/c spleen cells, are added to each well.
  • the growth medium used consisted of RPM1-1640 medium, 10% heat-inactivated fetal bovine serum (FBS) (Hyclone), 0.1 mM MEM nonessential amino acids, 1 mM sodium pyruvate, 2mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin (GIBCO Laboratories) and, for selection, hypoxanthine-aminopterin-thymidine (HAT) (Boehringer Mannheim).
  • FBS heat-inactivated fetal bovine serum
  • HAT hypoxanthine-aminopterin-thymidine
  • a solid-phase radioimmunoassay is employed for screening supernatants for the presence of mAbs specific for rhTNF ⁇ fragments including portions of residues 59-80, 87-108 or both 59-80 and 87-108 of hTNF ⁇ (of SEQ ID NO:1). This assay is described in Example II, above. The background binding in this assay is about 500 cpm. A supernatant is considered positive if it yielded binding of 2000 cpm or higher.
  • one or more positive supernatants are routinely identified by RIA. Of these positive supernatants, the highest binding (as shown by the higher cpm values) are subcloned at limiting dilution on mouse feeder cells. Upon further analysis of the supernatants in neutralization assays, routinely one or more antibodies are found to have potent inhibiting and/or neutralizing activity. These positive and inhibiting and/or neutralizing hybridoma lines are then selected and maintained in RPM1-1640 medium with 10% FBS (GIBCO), 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
  • GEBCO FBS
  • Murine and chimeric antibodies, fragments and regions are obtained by construction of chimeric expression vectors encoding the mouse variable region of antibodies obtained in Example XIV and human constant regions, as presented in Examples IV-IX above.
  • the resulting chimeric A2 antibody is purified from tissue culture supernatant by Protein A-Sepharose chromatography. The supernatant is adjusted to 0.1M Tris, 0.002M EDTA, pH 8.0 and loaded on a Protein A-Sepharose column equilibrated in the same buffer. The IgG is then eluted with 0.1M citrate, pH 3.5, neutralized with 1M Tris, and dialyzed into phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the purified murine and chimeric antibodies, fragments and regions are evaluated for its binding and inhibiting and/or neutralizing activity.
  • Both the murine and chimeric anti-TNF ⁇ antibodies of the present invention are determined to have potent TNF-inhibiting and/or neutralizing activity, as shown for example, in the TNF cytotoxicity assay described above, expressed as the 50% inhibitory Dose (ID50).
  • TNF-1, TNF-2 and TNF-3 three other murine anti-TNF ⁇ antibodies (termed TNF-1, TNF-2 and TNF-3) of comparable binding affinity to TNF are found to have ID50 values of 1-2 orders of magnitude greater, and thus have significantly less potent in neutralization, than both the murine and chimeric anti-TNF ⁇ antibodies of the present invention.
  • both the murine and chimeric anti-TNF ⁇ antibodies of the present invention inhibited or neutralized both natural and rhTNF in a dose-dependent manner in the cytotoxicity assay.
  • Such inhibiting and/or neutralizing potency at antibody levels below 1 ⁇ g/ml, can easily be attained in the blood of a subject to whom the antibody is administered.
  • highly potent inhibiting and/or neutralizing anti-TNF antibodies, in particular the chimeric antibody are preferred for therapeutic use in TNF ⁇ -mediated pathologies or conditions.
  • the ability of cA2 to inhibit or neutralize TNF-induced IL-6 secretion is evaluated using cultured human diploid FS-4 fibroblasts.
  • TNF procoagulant and adhesion molecule activities of endothelial cells
  • EC endothelial cells
  • this may be associated with the vascular damage, disseminated intravascular coagulation, and severe hypotension that is associated with the sepsis syndrome. Therefore, the ability of both the murine and chimeric anti-TNF ⁇ antibodies of the present invention, as obtained according to Examples XIV and XV, to block TNF-induced activation of cultured human umbilical vein endothelial cells (HUVEC) is evaluated.
  • TNF stimulation of procoagulant activity is determined by exposing intact cultured HUVEC cells to TNF (with or without antibody) for 4 hours and analyzing a cell lysate in a human plasma clotting assay. The results are expected to show the expected upregulation by TNF of HUVEC procoagulant activity (reflected by a decreased clotting time).
  • Both the murine and chimeric anti-TNF ⁇ antibodies of the present invention as obtained according to Examples XIV and XV, are expected to effectively inhibit or neutralize this TNF activity in a dose-dependent manner.
  • TNF In addition to stimulating procoagulant activity, TNF also induces surface expression of endothelial cell adhesion molecules such as ELRM-1 and ICAM-1.
  • endothelial cell adhesion molecules such as ELRM-1 and ICAM-1.
  • the ability of both the murine and chimeric anti-TNF ⁇ antibodies of the present invention, as obtained according to Examples XIV and XV, are expected to inhibit or neutralize this activity of TNF is measured using an ELAM-1 specific detection radioimmunoassay.
  • Cultured HUVEC are stimulated with 250 ng/ml rhTNF (Dainippon, Osaka, Japan) with or without antibody at 37°C overnight in a 96-well plate format.
  • Surface expression of BLAM-1 is determined by sequential addition of a mouse anti-human ELBM-1 mAb and 125 I-labelled rabbit anti-mouse immunoglobulin second antibody directly to culture plates at 4°C.
  • TNF is expected to induce the expression of BLAM-1 on the surface of cultured HUVEC cells, and this activity is again expected to be effectively blocked in a dose-related manner by both the murine and chimeric anti-TNF ⁇ antibodies of the present invention, as obtained according to Examples XIV and XV.
  • TNF is known to stimulate mitogenic activity in cultured fibroblasts.
  • Both the murine and chimeric anti-TNF ⁇ antibodies of the present invention are expected to inhibit or neutralize TNF-induced mitogenesis of human diploid FS-4 fibroblasts cultures, confirming the potent inhibiting and/or neutralizing capability of both the murine and chimeric anti-TNF ⁇ antibodies of the present invention, as obtained according to Examples XIV and XV against a broad spectrum of in vitro TNF biological activities.
  • both the murine and chimeric anti-TNF ⁇ antibodies of the present invention are expected to reduce mortality to 0-30 percent with 0.4 mg/kg of antibody, and to 0-10 percent with 20 mg/kgs.
  • both the murine and chimeric anti-TNF ⁇ antibodies of the present invention are capable of inhibiting and/or neutralizing the biological activity of TNF in vivo as well as in vitro .
  • Chimeric IgG1 anti-human TNF monoclonal antibody cA2 was administered to healthy male human volunteers as patients. One hour after receiving 4 ng/kg of an NIH reference endotoxin, the volunteers were administered either saline, as a control, or 0.01, 0.10 or 10 mg/kg of cA2 in a pharmaceutically acceptable form. TNF levels in serum were measured over time and were found to show a dose dependent decrease in peak TNF levels with no TNF being detected in volunteers receiving a 10 mg/kg dose of cA2. Accordingly, therapy with an anti-TNF antibody of the present invention is expected to inhibit TNF-mediated effects in humans.

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